Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus
Ianniciello, G.; Masullo, M.; Gallo, M.; Arcari, P.; Bocchini, V.
Biotechnology and Applied Biochemistry 23(1): 41-45
1996
ISSN/ISBN: 0885-4513 PMID: 8867895 Document Number: 469240
The elongation factor 1-alpha from the archaeon Sulfolobus solfataricus (SsEF-1-alpha) was expressed in Escherichia coli and purified. The SsEF- 1-alpha gene was amplified by PCR and cloned in the Ndel site of the pT7-7 expression vector, under the control of the promoter of T7 RNA polymerase. Upon induction with isopropyl beta-D-thiogalactopyranoside, the recombinant SsEF-1-alpha (recSsEF-1-alpha) was purified from the E. coli S-100 extract by a two-step procedure. From 1 litre of cell culture, about 2 mg of purified recSsEF-1-alpha was obtained. The N-terminal sequence of the first 30 amino acid residues of recSsEF-1-alpha was identical with that translated from the nucleotide sequence of the corresponding gene, except for the initial residue, which in recSsEF-1-alpha was Ser instead of Met. The M-r of recSsEF-1-alpha (determined by electrospray MS) was almost coincident with that of the naturally occurring SsEF-1-alpha (SsEF-1-alpha). The thermal-inactivation and thermophilicity profiles of SsEF-1-alpha and recSsEF-1-alpha were identical. Concerning the functional properties, recSsEF-1-alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to elicit an NaCl-dependent GTPase activity (Masullo, De Vendittis and Bocchini (1994) J. Biol. Chem. 269, 20376-20379) with the same efficiency as that displayed by SsEF-1-alpha.